The biochemistry of the site-specific recombination that integrates the genome of bacteriophage lambda into that of its E. coli host has been studied. A novel assay has been developed which is based on restriction endonuclease cleavage of parental and recombinant DNA followed by agarose gel electrophoresis. It has been found that all steps of the breakage and reunion cycle of recombination are completed in vitro. Furthermore, closed circular DNA is found to be the only efficient substrate. This finding and the observation that product circular DNA is catenated suggest that supertwisted DNA is the preferred substrate for the recombination reaction.